Telomeres consist of protein-bound DNA repeats which protect the chromosome ends. Telomeres are maintained by the enzyme telomerase, which is minimally composed of a protein reverse transcriptase and a template-containing RNA. Most cancer and stem/progenitor cells rely on telomerase to support continued cell division. In these cells, telomerase maintains telomeres by replenishing the terminal DNA repeats lost with each cell division, thereby avoiding the senescence and cell death caused by critically short telomeres. Because most tumors have high levels of telomerase while most somatic cells do not, telomerase represents a promising target for anticancer therapy. The laboratory of Dr. Elizabeth Blackburn pioneered the development of a novel telomerase-targeting strategy that employs mutant forms of the telomerase RNA (termed MT-hTers) that contain specific template mutations. Overexpression of these MT-hTersin cancer cells leads to incorporation of mutant telomeric repeats, disruption of the protective telomeric caps, and induction of a DNA damage response that inhibits cell propagation and promotes apoptosis. The work described in this proposal will investigate several critical aspects of MT-hTer function. First, cell culture and mouse xenograft systems will be used to identify the cellular pathways which mediate the anticancer effects of two different MT-hTers, knowledge of which will allow us to understand and ultimately predict the response of different cancers to this therapy. Second, the impact of MT-hTer treatment on telomerase-positive adult stem cells will be evaluated through studies with human hematopoietic stem and progenitor cells. Together, these experiments will provide important insight into maximizing the anticancer effects of MT-hTer while minimizing unwanted side effects. In addition, this work will map dysfunctional telomere response pathways in several clinically-relevant cell types. This five-year proposal also includes a career development plan designed to prepare Dr. Bradley Stohr for an academic research career. By completing the above research studies, participating in didactic training, and interacting with a carefully chosen team of advisors, consultants, and collaborators, Dr. Stohr will obtain the technical and intellectual expertise necessary for the transition to independence.